The present invention relates to the field of paramyxoviridae vaccines and is particularly concerned with vaccines comprising DNA encoding the fusion (F) protein of respiratory syncytial virus (RSV) in an alphavirus vector.
Human respiratory syncytial virus (RSV) has been identified as a major pathogen responsible for severe respiratory tract infections in infants, young children and the institutionalized elderly (refs. 1, 2, 3, 4xe2x80x94throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Global mortality and morbidity figures indicate that there is an urgent need for an efficacious RSV vaccine (refs. 5, 6). In the USA alone, approximately 100,000 children are hospitalized annually with severe cases of pneumonia and bronchiolitis resulting from an RSV infection. Inpatient and ambulatory care for children with RSV infections has been estimated to cost in excess of $340 million each year in the USA. The World Health Organization (WHO) and the National Institute of Allergy and Infectious Disease (NIAID) vaccine advisory committees have ranked RSV second only to HIV for vaccine development. Both the annual morbidity and mortality figures as well as the staggering health care costs for managing RSV infections have provided the incentive for aggressively pursuing the development of efficacious RSV vaccines. However, such a vaccine is still not available.
Formalin-inactivated (FI-RSV) and live attenuated RSV vaccines have failed to demonstrate efficacy in clinical trials (refs. 7, 8, 9, 10). Moreover, the formalin-inactivated RSV vaccine caused enhanced disease in some children following exposure to wild-type RSV (refs. 7, 8, 9, 10). Elucidation of the mechanism(s) involved in the potentiation of RSV disease is important for the design of safe RSV vaccines, especially for the seronegative population. Recent experimental evidence suggests that an imbalance in cell-mediated responses may contribute to immunopotentiation. Enhanced histopathology observed in mice that were immunized with the FI-RSV and challenged with virus could be abrogated by depletion of CD4+ cells or both interleukin-4 (IL-4) and IL-10.
The RSV fusion (F) glycoprotein is one of the major immunogenic proteins of the virus. This envelope glycoprotein mediates both fusion of the virus to the host cell membrane and cell-to-cell spread of the virus (ref. 1). The F protein is synthesized as a precursor (F0) molecule which is proteolytically cleaved to form a disulphide-linked dimer composed of the N-terminal F2 and C-terminal F1 moieties (ref. 11). The amino acid sequence of the F protein is highly conserved among RSV subgroups A and B and is a cross-protective antigen (refs. 6, 12). In the baculovirus expression system, a truncated secreted version of the RSV F protein has been expressed in Trichoplusia ni insect cells (ref. 13). The recombinant protein was demonstrated to be protective in the cotton rats (ref. 13).
Studies on the development of live viral vaccines and glycoprotein subunit vaccines against parainfluenza virus infection are being pursued. Clinical trial results with a formalin-inactivated PIV types 1, 2, 3 vaccine demonstrated that this vaccine was not efficacious (refs. 14, 15, 16). Further development of chemically-inactivated vaccines was discontinued after clinical trials with a formalin-inactivated RSV vaccine demonstrated that not only was the vaccine not effective in preventing RSV infection but many of the vaccinees who later become infected with RSV suffered a more serious disease. Most of parainfluenza vaccine research has focused on candidate PIV-3 vaccines (ref. 17) with significantly less work being reported for PIV-1 and PIV-2. Recent approaches to PIV-3 vaccines have included the use of the closely related bovine parainfluenza virus type 3 and the generation of attenuated viruses by cold-adaptation of the virus (refs. 18, 19, 20, 21).
Another approach to parainfluenza virus type 3 vaccine development is a subunit approach focusing on the surface glycoproteins hemagglutinin-neuraminidase (HN) and the fusion (F) protein (refs. 22, 23, 24). The HN antigen, a typical type II glycoprotein, exhibits both haemagglutination and neuraminidase activities and is responsible for the attachment of the virus to sialic acid containing host cell receptors. The type I F glycoprotein mediates fusion of the viral envelope with the cell membrane as well as cell to cell spread of the virus. It has recently been demonstrated that both the HN and F glycoproteins are required for membrane fusion. The F glycoprotein is synthesized as an inactive precursor (F) which is proteolytically cleaved into disulfide-linked F2 and F1 moieties. While the HN and F proteins of PIV-1, -2 and -3 are structurally similar, they are antigenically distinct. Neutralizing antibodies against the HN and F proteins of one of PIV type are not cross-protective. Thus, an effective PIV subunit vaccine must contain the HN and F glycoproteins from the three different types of parainfluenza viruses. Antibody to either glycoprotein is neutralizing in vitro. A direct correlation has been observed between the level of neutralizing antibody titers and resistance to PIV-3 infections in infants. Native subunit vaccines for parainfluenza virus type 3 have investigated the protectiveness of the two surface glycoproteins. Typically, the glycoproteins are extracted from virus using non-ionic detergents and further purified using lectin affinity or immunoaffinity chromatographic methods. However, neither of these techniques may be entirely suitable for large scale production of vaccines under all circumstances. In small animal protection models (hamsters and cotton rats), immunization with the glycoproteins was demonstrated to prevent infection with live PIV-3 (refs. 25, 26, 27, 28, 29).
The HN and F glycoproteins of PIV-3 have also been produced using recombinant DNA technology. HN and F glycoproteins have been produced in insect cells using the baculovirus expression system and by use of vaccinia virus and adenovirus recombinants (refs. 30, 31, 32, 33, 34). In the baculovirus expression system, both full-length and truncated forms of the PIV-3 glycoproteins as well as a chimeric F-HN fusion protein have been expressed. The recombinant proteins have been demonstrated to be protective in small animal models (see WO91/00104, U.S. application Ser. No. 07/773,949 filed Nov. 29, 1991, assigned to the assignee hereof).
Semliki Forest virus (SFV) is a member of the Alphavirus genus in the Togaviridae family. The mature virus particle contains a single copy of a ssRNA genome with a positive polarity that is 5xe2x80x2-capped and 3xe2x80x2-polyadenylated. It functions as an mRNA and naked RNA can start an infection when introduced into cells. Upon infection/transfection, the 5xe2x80x2 two-thirds of the genome is translated into a polyprotein that is processed into the four nonstructural proteins (nsP1 to 4) by self cleavage. Once the ns proteins have been synthesized they are responsible for replicating the plus-strand (42S) genome into full-length minus strands (ref. 14). These minus-strands then serve as templates for the synthesis of new plus-strand (42S) genomes and the 26S subgenomic mRNA (ref. 14). This subgenomic mRNA, which is colinear with the last one-third of the genome, encodes the SFV structural proteins.
In 1991 Liljestrom and Garoff (ref. 15) designed a series of expression vectors based on the SFV cDNA replicon. These vectors had the virus structural protein genes deleted to make the way for heterologous inserts, but preserved the nonstructural coding region for production of the nsPl to 4 replicase complex. Short 5xe2x80x2 and 3xe2x80x2 sequence elements required for RNA replication were also preserved. A polylinker site was inserted downstream from the 26S promoter followed by translation stop sites in all three frames. An SpeI site was inserted just after the 3xe2x80x2 end of the SFV cDNA for linearization of the plasmid for use in vitro transcription reactions.
Injection of SFV RNA encoding a heterologous protein have been shown to result in the expression of the foreign protein and the induction of antibody in a number of studies (refs. 16, 17). The use of SFV RNA inoculation to express foreign proteins for the purpose of immunization would have several of the advantages associated with plasmid DNA immunization. For example, SFV RNA encoding a viral antigen may be introduced in the presence of antibody to that virus without a loss in potency due to neutralization by antibodies to the virus. Also, because the protein is expressed in vivo the protein should have the same conformation as the protein expressed by the virus itself. Therefore, concerns about conformational changes which could occur during protein purification leading to a loss in immunogenicity, protective epitopes and possibly immunopotentiation, could be avoided by plasmid DNA immunization.
In copending U.S. patent application Ser. No. 08/476,397 filed Jun. 7, 1995 (now U.S. Pat. No. 6,019,980), assigned to the assignee hereof and the disclosure of which is incorporated herein by reference (WO96/40945), there is described reference the use of plasmid vectors containing RSV F protein-encoding DNA for DNA immunization against RSV infection. In copending U.S. patent application Ser. No. 08/896,442 filed Jul. 18, 1997, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference, there is described the use of plasmid vectors containing RSV G protein-encoding DNA for DNA immunization against RSV infection.
In my copending U.S. patent application Ser. No. 08/923,558, filed Sep. 4, 1997 (now U.S. Pat. No. 6,060,308), assigned to the assignee hereof and the disclosure of which is incorporated by reference, I describe a DNA vector using an alphavirus vector, including Semliki Forest virus vector, containing a DNA sequence encoding a paramyxovirus protein, specifically RSV-F, for making an RNA transcript for immunization.
In WO95/27044, the disclosure of which is incorporated herein by reference, there is described the use of alphavirus CDNA vectors based on cDNA complementary to the alphavirus RNA sequence. Once transcribed from the CDNA under transcriptional control of a heterologous promoter, the alphavirus RNA is able to self-replicate by means of its own replicase and thereby amplify the copy number of the transcribed recombinant RNA molecules.
Infection with RSV leads to serious disease. It would be useful and desirable to provide improved vectors for in vivo administration of immunogenic preparations, including vaccines, for protection against disease caused by RSV and other paramyxoviruses. In particular, it would be desirable to provide vaccines that are immunogenic and protective in humans, including seronegative infants, that do not cause disease enhancement (immunopotentiation).
The present invention provides novel immunogenic materials and immunization procedures based on such novel materials for immunizing against disease caused by respiratory syncytial virus. In particular, the present invention is directed towards the provision of DNA vaccines against disease caused by infection with paramyxoviridae.
In accordance with one aspect of the present invention, there is provided a vector, comprising a first DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complement of complete alphavirus RNA genome replication regions to permit in vivo replication; a second DNA sequence encoding a paramyxovirus protein or a protein fragment that generates antibodies that specifically react with the paramyxovirus protein, the second DNA sequence being inserted into a region of the first DNA sequence which is non-essential for replication; the first and second DNA sequences being under transcriptional control of a promoter; and a third DNA sequence located adjacent the second DNA sequence to enhance the immunoprotective ability of the paramyxovirus protein when expressed in vivo from the vector in a host.
The paramyxovirus protein may be selected from the group consisting of a parainfluenza virus (PIV) and a respiratory syncytial virus (RSV). The PIV protein may be from PIV-1, PIV-2, PIV-3 or PIV-4, particularly the HN and F glycoproteins of PIV-3. The RSV protein particularly may be the F or G glycoprotein of RSV.
The second DNA sequence may encode a full length RSV F protein, or may encode a RSV F protein lacking the transmembrane anchor and cytoplasmic tail. The lack of the coding region for the transmembrane anchor and cytoplasmic tail results in a secreted form of the RSV F protein. Alternatively, as described in the aforementioned U.S. patent application Ser. No. 08/896,500, the second DNA sequence may encode the full-length RSV-G protein or a truncated RSV G protein lacking a transmembrane region, resulting in a secreted form of the protein.
The alphavirus preferably is a Semliki Forest virus and the first DNA sequence is the Semliki Forest viral sequence contained in plasmid PSFVI.
The third nucleotide sequence may comprise a pair of splice sites to prevent aberrant mRNA splicing, in vivo, whereby substantially all transcribed mRNA from the vector upon administration encodes the RSV protein. Such third nucleotide sequence is preferably located between the first nucleotide sequence and the promoter sequence. Such third nucleotide sequence may be that of rabbit xcex2-globin intron II, as shown in FIG. 8 of copending U.S. patent application Ser. No. 08/476,397 (WO 96/040945).
The promoter sequence may be an immediate early cytomegalovirus (CMV) promoter. The human cytomegalovirus Intron A sequence may be provided downstream of the promoter and upstream of the third nucleotide sequence.
A vector encoding the F protein and provided in accordance with one embodiment of the invention may be specifically pMP44, having the identifying characteristics shown in FIG. 1D.
The vectors provided herein may be used to immunize a host against RSV infection or disease by in vivo expression of RSV F protein or RSV G protein, which may lack a transmembrane region, or other paramyxovirus protein, following administration of the vectors. In accordance with a further aspect of the present invention, therefore, there is provided a method of immunizing a host against disease caused by infection with respiratory syncytial virus or other paramyxovirus, which comprises administering to the host an effective amount of a vector provided herein.
The present invention also includes a novel method of using a gene encoding an RSV F or G protein or a fragment of an RSV or G protein capable of generating antibodies which specifically react with RSV F or G protein to protect a host against disease caused by infection with respiratory syncytial virus, which comprises isolating the gene; operatively linking said gene to a DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complement of complete alphavirus RNA genome replication regions in a region of said DNA sequence which is non-essential for replication to form a vector wherein said gene and DNA sequence are under transcriptional control of a promoter; operatively linking the gene to an immunoprotection enhancing sequence to produce an enhanced immunoprotection by the RSV F or G protein in the host, preferably by introducing the immunoprotection enhancing sequence between the control sequence and the alphavirus sequence; and introducing the vector into the host. A corresponding procedure may be used for other paramyxoviridae.
In addition, the present invention includes a method of producing a vaccine for protection of a host against disease caused by infection with respiratory syncytial virus (RSV), which comprises isolating a first DNA sequence encoding an RSV or G protein, from which the transmembrane anchor and cytoplasmic tail may be absent; operatively linking said first DNA sequence to a second DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complete alphavirus genome replication regions in a region of said second DNA sequence which is non-essential for replication to form a vector wherein said first and second DNA sequences are under transcriptional control of a promoter; operatively linking the first nucleotide sequence to a third nucleotide sequence to enhance the immunoprotective ability of the RSV F or G protein when expressed in vivo from the vector in a host; and formulating the vector as a vaccine for in vivo administration. A corresponding procedure may be used for other paramyxoviridae.
The present invention further includes a vaccine for administration to a host, including a human host, produced by the method as well as immunogenic compositions comprising an immunoeffective amount of the vectors described herein.